Aerobic fermentation apparatus

ABSTRACT

Yeasts are grown aseptically by an improved aerobic fermentation process, employing an aqueous ethanolic substrate fortified with nutrient elements, preferably under oxygen-limited conditions. Fermentation is effected in a continuous manner in a zone maintained under super-atmospheric pressure. Micro-nutrients are added to the substrate separately from macronutrients as a sterile aqueous solution, preferably containing iron as ferric citrate. A preferred yeast is Candida utilis.

ilnited States Patent Ridgway, Jr. et al.

[ Nov. 12, 1974 AEROBIC FERMENTATION APPARATUS Inventors: John A. Ridgway, Jr., LaPorte, lnd.;

Terry A. Lappin, Palos Heights; Benny Moses Benjamin, Chicago, both of 111.; Joseph B. Corns, Munster, lnd.; Cavit Akin, Oakbrook, lll.

Assignee: Standard Oil Company, Chicago, 111.

Filed: Mar. 7, 1973 Appl. No.: 338,751

Related US. Application Data Division of Ser. No. 85,334, m. 30, 1970,

abandoned.

U.S. Cl 195/142, 195/143, 195/139 Int. Cl Cl2b l/00 Field of Search 195/142, 143, 139, 127;

References Cited UNITED STATES PATENTS /1969 Freedman et a1 195/142 2,312,217 2/1943 King et a1. /175 3,681,200 8/1972 Ridgway. .lr. /142 3,326,279 6/1967 'Eisberg et a1. 165/173 Primary ExaminerAlvin E. Tanenholtz Attorney, Agent, or Firm-Werten F. W. Bellamy; Arthur G. Gilkcs; William T. McClain [57] ABSTRACT Yeasts are grown aseptically by an improved aerobic fermentation process, employing an aqueous ethanolic substrate fortified with nutrient elements, preferably under oxygen-limited conditions. Fermentation is effected in a continuous manner in a zone maintained under super-atmospheric pressure. Micro-nutrients are added to the substrate separately from macronutrients as a sterile aqueous solution, preferably containing iron as ferric citrate. A preferred yeast is Candida utilis.

3 Claims, 4 Drawing Figures F E RME N 7' 0/? Lg r/OEG 1 AEROBIC FERMENTATION APPARATUS This is a division, of application Ser. No. 85,334, filed Oct. 30, 1970 and now abandoned.

BACKGROUND OF THE INVENTION Recent concern for the welfare of the world population has included consideration of additional means for feeding the repidly increasing number of people involved. The problem embraces providing both adequate per capita caloric intake and a balanced diet, with particular reference to the acknowledged lack of sufficient protein-affording-- foods in many parts of the world. One means for providing necessary protein supplies is through the growthof single-cell proteinaffording microorganisms, such as yeasts, bacteria and algae, for use as either foods or food supplements.

Production of single cell protein (SCP) materials in large quantity may be accomplished by fermentation processes employing, for example, carbohydrate, hydrocarbon or oxygenated hydrocarbon materials as substrate. Principal requirements are that the substrate material be inexpensive and readily consumed by the selected microorganism so that process costs are not excessive. Equally important is the acceptability and utility of the SCP material as a food or food component. The latter considerations include taste and odor factors relating to public acceptance as well as metabolic and toxicity factors relating to suitability of SCP material for inclusion in the human diet.

Both the technical and the patent literature describe fermentation processes for production of microorganisms which readily afford useful SCP materials. For example, yeasts have been grown on the polysaccharides contained in waste sulfite liquor, the normal alkane components of a gas oil hydrocarbon fuel, and on a mixture of oxygenated hydrocarbons. Production of bacteria has been similarly described. Fermentation to produce yeasts or bacteria comprises an oxidation process, evolving much heat and requiring both substantial oxygen transfer and good control of fermentation temperature. Preferred substrate materials will already contain as much combined oxygen as possible in order to minimize the heat release and the oxygen requirement. Production of food-grade SCP material may also require an extraction step to limit the presence of undesirable, residual substrate material such as highmolecular-weight hydrocarbons or slowly fermented oxygenated hydrocarbon species.

Most of the fermentation processes planned or in use currently for production of SCP material are intended to provide primarily an animal feed supplement and hence to suply protein forhuman consumption only indirectly. However, certain microorganisms, notably yeasts within the Saccharomycetoideae and Cryptococcoideae sub-families, have been certified by the Food and Drug Administration for direct use in foods intended for human consumption.

One highly desirable substrate material is ethanol. It exhibits complete water solubility, is already in a partially-oxidized state, is itself acceptable for use in foods, and creates no problem as to removal from the produced microorganism cells. However, ethanol is a growth inhibitor. to many microorganisms and some others do not grow wellin its presence.

The economics of SCP production require that the substrate material be relatively inexpensive. In comparexpensive to require that it be used most efficiently if selected to serve as a substrate.

SUMMARY OF THE INVENTION It is the purpose of this invention to provide an improved e c srmen at c Pro e s an appa atus fo the continuous production of food yeasts.

, Another object of this invention is to employ optimally the inorganic nutrient materials necessary to the growth of the desired yeasts.

It is a further object of this invention to provide an economic source of high-quality protein material, for use as a food or food ingredient intended for human consumptiomby utilizing the growth of a yeast having FDA approval for use in foods on a substrate possessing a high proportion of combined oxygen and being itself of acceptable food quality. Specifically, a food yeast such as Candida utilis (Torula yeast) is grown on an ethanol substrate under conditions selected for maximum conversion of the substrate to a useful protein product.

Further objects will be evident from the following description of the invention.

DESCRIPTION OF THE DRAWINGS The attached drawings are illustrative of typical embodiments of this invention.

FIG. 1 illustratesa flow process pattern. FIG. 2 presents illustrative data in chart form. FIGS. 3 and 4 describe an embodiment of apparatus particularly applicable to the practice of this invention.

FIG. 1 presents in schematic fashion one embodiment of the invention, comprising a yeast production process operating continuously under steady-state conditions employing, ethanol. as a substrate.

In this embodiment of the invention mixer 14 is fed with ethanol through, line 11, a concentrated solution of macronutrients through line 12, and make-up water through line 13. Nutrients contained in at least a portion of the liquid fermentor effluent are also fed to mixer 14 through. line 52,. After thorough mixing with stirrer 15 the solution is pumped. by means not shown through line. 16, preheater 17, and line 18 to sterilizer 19. Steam is supplied to the sterilizerthrough coil 20. Optionally steam may be injected directly into line 18 by means not shown. After sterilization the solution is passed through, line 2 1, preheater 17, line 22, cooler line 24 .P essure du. va ZA a line. .5 to r r. 40- The m c onutient ncludi s rqn citrate, are supplied, as, an aqueous. concentrate through line 36 sterilizer 37; and line;3 .9. Steamfor the sterilizer is supplied throughcoil38 Air, iscontinuously taken in through line 26 tocompressor 27: and the compressed air stream is thenpassedthrough lines.,28 and 2 9, st erilizing filter S0, and line 31: to air sparger 32 situated in the bottom sectionof fermentor40, Ammonia from liquid storage is vaporized, andpassed through lines 33 and 34 into the compressed air stream so that the airammonia. mixture is carried through line 29, filter30 n line. 31 to Par =3 .;-.0P icmal y h p ed ammonia stream may. be f ed tof ermentor 40 by means of lines 35 andZS.

The continuoussupply of substrate, nutrients and oxygen effects a continuous multiplication of yeast cells (originally added as a batchstarter culture prepared in equipment not shown) within fermentor 40. Agitation of the gas-liquid-solid mixture is accomplished with agitator 44 and sparger 32. Metabolism of the ethanol by yeast generates a substantial amount of heat so that a coolant is circulated through line 41, cooling coil 43-43 and line 42.

Fermentation broth is continuously removed from fermentor 40 through bottom draw-off line 45 to separator 46 where most of the liquid, containing residual substrate and nutrient material, is separated from the yeast cells. The separated liquid phase is removed from the separator through line 51 and either returned to mixer 14 through line 52 or sent to waste disposal through line 53. The product yeast cells, in the form of a cream, are removed from the separator through line 47, optionally washed in equipment not shown, and sent to drier 48. Removed water leaves the drier through line 49 and the dried food yeast product is taken through line 50 to cooling and storage.

The spent air stream, containing product carbon dioxide, is continuously removed from fermentor 40 through line 54, pressure-responsive automatic valve 55 and line 56. Line 56 transmits the spent air to an appropriate stack, or, when the spent air contains a significant amount of entrained ethanol, to a wtaer scrubber, not shown. Water from the scrubber, containing recovered ethanol, may serve as make-up water supplied through line 13 to mixer 14.

FIG. 2 presents a graphic illustration of results obtained employing a particularly advantageous mode of practicing the invention. It is discussed further in relation to Example V and Table III.

FIG. 3 presents an elevational view partly in crosssection setting forth details of an eminently suitable internal cooling apparatus for use in the fermentation process of this invention.

The embodiment of the bird-cage cooling apparatus shown in FIG. 3 is contained within fermentor vessel 101 which is conventionally fitted with nutrient inlet 102, air inlet line 103, air inlet extension line 104, air sparger 105, baffles 106 and 106a, agitator 107 connected by shaft 108 to external drive means not shown, fermentation broth outlet 109 and air outlet 110.

The cooling apparatus comprises horizontal circular header pipes 120 and 121, having substantially the same mean diameter, joined together by a plurality of vertical connector pipes illustrated by 122 and 122a, arranged in two parallel rows around the circumference of header pipes 120 and 121. The connector pipes 122, 122a have a diameter no greater than about 1/3 that of the header pipes and extend roughly threefourths the length of fermentor vessel 101. The bottom header pipe 120 is attached to coolant inlet line 123 and upper header pipe 121 is attached to coolant exit line 124, both of lines 123 and 124 sealably extending through the wall of fermentor vessel 101.

In practice liquid ammonia coolant enters header pipe 120 through line 123 and is distributed to connector pipes 122, 122a which provide surface for heat transfer with the agitated fermentation broth contained in fermentor vessel 101. The ammonia vaporizes as it absorbs heat from the broth, passing as a mixture of liquid and vapor into header pipe 121 and coolant exit line 124. The vapor-liquid mixture is then separated, the vapor stream is compressed by means not shown, and the resulting liquid is added to the separated liquid DESCRIPTION OF THE INVENTION This invention embraces the aseptic growth of selected yeasts on an ethanol substrate in a continuous manner, providing for optimized utilization of selected nutrient elements, including iron. The choice of ethanol as the sole source of carbon in the substrate eliminates substantially problems relating to production of protein material suitable for direct human consumption. Ethanol is readily available and accepted as a foodstuff. Its fermentation products will not contain toxic residual substrate. Its volatility assures that residual ethanol will be removed readily during drying of the microorganism product. Its solubility in water obviates I multi-phase physical problems present with polysaccharide or hydrocarbon substrate materials.

Many microorganisms do not grow on alcoholic substrates. Suitable yeasts which do metabolize ethanol include those listed in Table I.

TABLE I SUITABLE YEASTS FOR USE WITH ETHANOL SUBSTRATE C apsularir Fibulicer .Iavanensir Cerevisiae do. F mgilis do. Lat'lis Hansenula Anomala M [so M rakii F arinosa F ermcnluns U lilis Guilliermondii E ndom ycopris do. S acrha mm yces Preferred yeasts include Saccharomyces cerevisiae, Saccharomyces fragilis, and Candida utilis. These are preferred because they already possess FDA approval for use in foods intended for human consumption.

Aerobic growth of the selected yeast is effected on a large scale in a continuous, aseptic fermentation process wherein sterile substrate, nutrients and oxygen are introduced continuously into a fermentor vessel while fermentation broth is continuously removed. Rapid exponential phase growth is maintained by control of the dilution rate (space velocity) through controlled addition of water to the fermentor. Suitable control devices are employed to maintain substantially steady-state conditions. Where the scale of yeast production is sufficiently large, it may be desirable to employ a plurality of fermentors in parallel arrangement. This affords better control of the fermentation while minimizing the shutdown costs should a fermentor become fouled in any manner. Fermentor effluents may thereafter be combined for subsequent downstream processing.

Nutrient Element Within the fermentation zone ethanol is maintained as an aqueous substrate having a concentration in the range from 50 to 3000 ppm, preferably from 100 to 500 ppm and most preferably about 200 ppm. lnorganic nutrients are maintained in the fermentation broth by continuous addition of aqueous solutions containing the nutrient elements to provide the ratios shown in Table ll.

TABLE II lNORGANlC NUTRIENTS lN FERMENTATION BROTH Typical Compound Nutrient Element Input, wt./l g. cells produced and sterilized by filtrationthrough a series of smallpore or membrane-type glass fiber'filters. When mixed with ammonia the mixed gases are passed through the Broad Range Preferred Range MACRO- NUTRIENTS Phosphorus H3PO4 l-5 g. 2-4 g.

Potassium KCL l5 do. 2-3 do. Magnesium MgCl,-6H O;MgSO 0.2-l do. 0.3-0.6 g. Calcium CaCl', 0001-] g. 0.00l0.2 g. Sodium Na CO -H O;NaCl 0.0l-0.l g. 0.0l-0.2 do. MICRO- NUTRIENTS lron Fe[C;,H,,(OH)(COO) 1-40 mg 6-l3 mg. Manganese MnSO H O l-20 do 4-8 do.

Zinc ZnSO -7H O 0.5-20 do. 2-6 mg. Molybdenum Na MoO -2H O 0.l-l0 do. 1-2 do. lodine Kl 0.1-10 do. 1-3 do. Copper CuSO '5H O 0.01-10 0.5-] do.

During the fermentation process ethanol is consumed the fermentor and all lines intended to pass sterile with evolution of carbon dioxide gas and an increase in streams be treated forabout minutes with steam at the acidity of the fermentation medium. Nitrogen is esa temperature of about 250F. sential to the growth of the microorganisms and is conln starting a fermentation, an initial loading of the veniently added to the fermentation broth as either anfermentor with aqueous substrate, ammonia and nutrihydrous or aqueous ammonia. Being an alkaline reaent elements is followed by innoculation of this aquegent, the addition of nitrogen as ammonia also serves ous medium with a culture of the selected yeast. Air is to decrease acidity in the fermentation broth. The p then sparged into the fermentor, usually with additional of the medium 15 mamtam d n her ng from to mechanical agitation provided. The fermentation zone pref r y fI'Om t0 d most p r ly at 15 maintained at a temperature 1n the range from 80 to about 4.0. This pH control 15 achieved by controlled 11()F and preferably about 90 to 100F., while the addition of ammonia- V p H n y top pressure is maintained within the range from 2 to The added inorganic nutrients are effective in pro- 40 20 R- -gi P l fflbout 10 P; gto assist in p moting yeast growth'only to the extent of their solubilserving aseptic conditions. The initial slow growth of ity in the fermentation broth. The requirement for the yeast is superseded after a few hours by the rapid phosphorus is customarily satisfied by addition of a exponential growth which is thereafter maintained in phosphate salt or phosphoric acid. When iron is also the fermentor by withdrawal of fermentation broth, required in the aqueous mixture of nutrients iron phoscomprising aqueous medium and suspended cell prodhate reci itates makin iron less available to the feruct, at a rate selected to maintain a cell concentration p p Ih b f dtht h f 15 ll mentation process. t as now een oun a an 1min t e range rom to wt. percent, genera y proved rate of yeast growth can be achieved by sepaabove about 2.0 wt. percent and preferably about 3.0 rately adding the micro-nutrient elements, including 50 wt. percent, suspended in the fermentation liquor. The iron, to the broth. Iron is preferably introduced as the withdrawal rate maintaining this cell concentration water-soluble salt of an organic polycarboxylic acid should provide an average residence time for fermentaand most preferably as iron citrate. This salt may be tion liquor in the fermentation zone in the range from formed i th aq s solution by th a i t ad. 2 to 4 hours and preferably about 3 hours. Etated indifditions of an inorganic iron salt, such as ferric chloride, fel'ent terms, the dilution rate should be m the range and i i id from 0.25 to 0.50/hr. and preferably should be about 0.33/hr.

hquld Streams are Sienhzed by bee-mug to a-b9ut Liquid level in the fermentor is maintained by addi- 300 F. under about 70 p.s.1.g. pressure prior to addition tion of aqueous ethanol, aqueous nutrients and ammoto the fermentor. Sterilization may also be accomh d b t t tion No Sterilization is nm to preserve the desired concentration levels and p y W g Jec t wh acidity. The withdrawn fermentation broth is sent to a g y require e ammomfi g i 3 separation stage, preferably a centrifuge, for recovery f ed as a ammonia g cfmvenllem y e mjecte of the cell product. The aqueous fermentation liquor Into the entering compresse air stream.-

discharged from the centrifugemay contain sufficient op ly enflched Wlth yg is compressed ethanol together with nutrient elements and ammonia to make this stream suitable for recycle. in a typical recycle operation about percent by volume of this stream is admixed with the continuously added streams after a suitable sterilization. The vol. percent discard serves to prevent buildup of less desirable inorganic ions such as chloride in the fermentation liquor.

The yeast cell product recovered from the separation zone may be washed with water, pressed and dried as required by the end use intended for the protein material.

Excess sterile compressed air is supplied to the fermentor after passage through a filtration zone. Oxygen utilization is usually in the range from to 60 percent of input and most frequently about 33 percent.

The concentration of dissolved oxygen in the fermentation liquor should be within the range from 0.1 to 0.3 ppm under oxygen-limiting conditions and may range as high as l to 4 ppm when operating under ethanollimiting conditions. Some foaming occurs in the fermentor but at the preferred low ethanol concentrations the foaming is not severe enough to require regular addition of an antifoam agent. Effluent air, containing product carbon dioxide, is exhausted from the fermentation zone through a pressure-responsive regulating valve, to maintain fermentor pressure and prevent entry of non-sterile materials which would contaminate the fermentor contents.

When the effluent gas stream contains a significant concentration of ethanol vapor it is desirable to pass this effluent stream through a water scrubber to recover the ethanol, later employing the ethanolic water as make-up to the fermentor.

The heat of fermentation is approximately 10,000 B.t.u./lb. cells so that temperature control within the range from 80 to 100F. requires extensive cooling. Where water is available at a sufficiently low temperature, cooling may be effected by once-through water circulation through cooling elements contained within the fermentation zone. In other circumstances a closed refrigeration system employing a refrigerant non-toxic to the system is preferred. Suitable refrigerants include ammonia and the Freons. With such refrigerants a small amount of leakage into the fermentor will present no adverse effects. Where ammonia is employed as the refrigerant, liquid ammonia is circulated through the cooling element where it boils, extracting heat evolved within the fermentation zone.

A particularly effective cooling element for use in any vertical cylindrical fermentor vessel and particularly with the process of this invention comprises two circular circumferential header tubes fitted horizontally within the fermentor and connected by a plurality of vertical cylindrical tubes, selected to have a total cross-section area equal to that of the top header tube. The cylindrical vertical tubes are arranged in parallel rows about the circumference of the header tubes to describe a bird-cage. Coolant circulates through the tubes which provide a large surface area for effective heat transfer.

Food industry practice should be followed in selecting equipment for use in the fermentation process of this invention. Type 304 stainless steel should be employed with foods or with liquids that eventually come in contact with food. Type 316 stainless steel is used where solutions contain high concentrations of chloride ion and where high temperatures are encountered. Whereever pressure and temperature requirements are not extreme, as in storage tanks, glass-fiber reinforced Nitrogen, total 9.2 wt. Nitrogen, protein 7.8 wt. Carbon 45.4 wt. Hydrogen 6.7 wt. 9% Phosphorus 2.0 wt. Ash 8.9 wt.

A typical amino acid profile and a typical vitamin content of Torula food yeast grown on ethanol are presented respectively in Tables Ill and IV.

Accordingly, a higher nutritive food or food ingredient comprising SCP material is made available by the practice of this invention.

EXAMPLES The following examples illustrate, without any implied limitation, the practice of this invention.

TABLE III AMINO ACID PROFILE OF ETHANOL GROWN TORULA YEAST of cell weight Lysine l-listidine Arginine Aspartic Acid Threoninc Serine Glutamic Acid Proline Glycine Alanine Cystine Valine Methionine lsoleucine Lcucine Tyrosine Phenylalanine Typtophane (Ammonia) TABLE IV TYPICAL VlTAMlN CONTENT OF ETHANOL GROWN TORULA YEAST Biotin Folic Acid lnositol Niacin Pantothenic Acid Panthenol P-Aminobenzoic Acid Riboflavin Vitamin B-6 Vitamin B-l2 ca Cholin Chloride Vitamin A EXAMPLEI In a 4-liter glass fermentor was placed 3 liters of aqueous mineral nutrient solution medium containing:

KH PO 1 o g./liter K H P 1.0 g./liter NH,Cl 1.0 g./liter MgSO, 1.0 g./liter CaCl 0.15 g./liter CuSO; H O 0 000] g./liter 0.0002 g./liter MmSO, H 0 0 0009 gJliter Na MQO, 2H,O 0 0004 g./liter ZnSO 7H O 0 0014 g./liter To this medium was added 30 ml. ethanol and FeCl (0.00l g./liter). Air was sparged in with agitation to obtain an oxygen absorption rate in the range from 100 to 140 millimoles/liter/hour. The fermentor temperature was maintained at 30C. and the pH was adjusted to 4.6 by addition of ammonia.

Freshly grown Torula yeast (Candida utilis) (50 ml. of 1 percent suspension) from a shaker culture was added to the fermentor. Cell growth was followed by measurement of optical denstiy during the aspecticbatch fermentation. Active cell growth started after about 3 hours and stopped when the cell concentration reached 0.7 g./100 ml suspension.

The product was harvested by centrifugation and dried at 100C. in an oven. The dry product was light brown in color and had a nutty'flavor.

EXAMPLE II The procedure of Example I was followed. When the cell concentration reached 0.6 g./l00 ml., mineral nutrient solution, ammonia, and ethanol were pumped into the fermentor at a steady rate while withdrawing fermentation broth to maintain a space velocity of 0.3/hr. Cell concentration in the effluent was maintained at 0.6-0.7 g./l00 ml.

EXAMPLE Ill Continuous growth of Candida utilis, A. T. C. C. No. 9256, was effected in a 28-liter fermentor vessel. After initial sterilization with steam the fermentor was loaded with an aqueous nutrient medium containing:

H PO (85%) 3.24 gJliter KOH 1.28 gJliter NaOH 0.02 g./liter MgSO, 1.30 gJliter CaCl,'2l-l 0 0.48 g./liter FeCl '6H O 1.55 mg./liter CuSO '5H O 0.10 mgjliter Kl 0.21 mg./liter MmSO 'H- O 1.84 mg./liter Na MoO -H-,O 0.41 mg./liter ZnSO,-7H,O 1.00 mg./liter Ethanol was added to provide a concentration of 0.2 wt. percent (2,000 ppm). Initially aqueous ammonia was added as a 30 percent solution in the amount of l ml./liter and was added thereafter as required to maintain pH 4.0 in the fermentor. An inoculum grown in a batch fermentor was added to provide a cell concentration of 0.1 g./100 ml. and allowed to grow at 90F. through several doubling cycles as in a batch run. Addition of nutrient solutin and ethanol and continuous medium. The iron (Fe was stabilized in aqueous off line was then begun and maintained at a dilution EXAMPLE IV The continuous run of Example III was repeated except for a separate addition of the nutrient element iron apart from other components of the aqueous nutrient solution as a complex with citric acid.

In continuous operation, employing this modified nutrient addition system, the cell concentration lined-out at 2.1 g./l00 ml. broth (ca. 2.1 wt. percent). The cell yield, based on ethanol consumed, was 80.1 wt. percent. The doubling time was 2.2 hours. Harvested dry cells were produced at a rate of 0.34 lb./hr./cu. ft. .fermentor volume.

EXAMPLE V TABLE V CELL GROWTH ON ETHANOL Dilution Rate, hr.- 0.33

0 -Limited Cell yield 83.8 84.0 82.8 71.5

N, wt. 8.7 8.9 9.1 9.2

Protein, wt. 45.0 44.6 47.3 46.7

EtOH-Limited Cell yield 71.6 72.1 70.3 65.6 62 5 N, wt. 8.9 9.3 9.1 9.1 9 6 Protein, wt. 46.8 46.2 45.7 44.4 47 2 "Wt. on ethanol consumed Calculated value (N-0.l53 X Nucleic Acid) X 6.25

EXAMPLE VI In the continuous production of Torula yeast (Candida utilis), grown on a substrate containing ethanol as the sole source of carbon, at the rate of 10,000 lbs./hr., there are provided six aerobic fermentors, each having a capacity of 50,000 gallons, arranged in two productiontrains. Each fermentor vessel has a diameter of 19 feet 'and a height of 25 feet. The use of several fermentors provide flexibility so that contamination of one fermentor still permits the plant to keep functioning.

In the process, ethanol vol. percent: 2188 gal./hr) is mixed with macro-nutrient salts, make-up water (9700 gal/hr.) and nutrient recycle in the manner described in FIG. I and the mixture continuously sterilized by heating with steam at 300F. The macronutrients are first dissolved in water to provide an aqueous concentrate which is then pumped to the mixing tank to provide the following feed rates for the component nutrients.

MACRONUTRIENTS Phosphoric Acid 80% Solution I780 lb./hr. Potassium Chloride KCl 558 do. Magnesium Chloride MgCl -6H,O 481 do. Calcium Chloride 21.5% Moisture 61 do. Ammonium Sulphate (NHJ SO, 62 do. Sodium Carbonate Na CO 'H O 31 do.

The micro-nutrients are similarly provided as an aqueous concentrate, sterilized and pumped directly to the fermentor to provide the following feed rates for the component nutrients.

MICRONUTRIENTS Ferric Citrate Fe[C;,H (OH)(COO) 16.0 lb./hr. Manganous Sulphate MnSO,-1-I,O 1.6 do. Zinc Sulphate ZnSO '7H,O 1.8 do. Sodium Molybdate Na M0O '2l-l O 0.3 do: Potassium Iodide Kl 0.2 do. Cupric Sulphate CuSO, 0.1

Cooled sterile liquid is sent to the continuous fermentors operated at 90F. under aseptic conditions. Air required for the fermentation is sterilized by passage through glass-fiber membrane-type filters and sparged into the bottom section of each fermentor where oxgen transfer is effected by intense agitation of the fermentation broth. Sterile air is introduced to each fermentor at a rate of 8,000 cu. ft./minute and oxygen transfer is effected with a turbine agitator. Anhydrous ammonia is added continuously to the fermentors as a nutrient, supplying the nitrogen content of the cells, in the total amount of 1,175 lbs/hr. which is sufficient to maintain the acidity level at pH 4.0.

Each fermentor is put on stream with addition of a starter culture of viable Candida utilis yeast cells grown in a batch seed tank.

Each fermentor is operated in the exponential phase of growth at 90F. and pH 4.0, effecting a doubling of cell weight every 2.1 hours. Residence time in the fermentors is 3 hours.

Spent air containing unabsorbed oxygen and product carbon dioxide is released through a regulating valve. The heat of fermentation is removed by passage of ammonia coolant through a bird-cage cooling element of the type shown schematically in FIGS. 3 and 4. The bird-cage cooling element comprises 2 circular headers, made of 2 feet diameter pipe, fitted within the fermentor vessel and having an outside diameter of 16 feet. The headers are connected by 380 vertical tubes 21 feet in length, having 1 inch diameter, arranged in two parallel rows about the circumference of the headers to provide 2,600 square feet of surface area.

Total fermentor effluent, amounting to 500,000 lb./hr. of fermentation broth containing 2.0 wt. percent yeast cells, is separated by centrifuging into a yeast cell cream (50,000 lb./hr. containing 20.0 wt. percent yeast cells) and a supernatant aqueous solution containing residual ethanol and nutrient elements. The supematant solution is partly (58,000 lb./hr.) sent to discard for waste processing and the remainder (392,000 lb./hr.) is recycled to the fermentor, in admixture with added nutrient elements and make-up water, after passing through the sterilizer.

The yeast cream is sent directly to a spray drier, recovered as a powder containing 5.0 wt. percent moisture, cooled and sent to storage.

What is claimed is:

1. In a continuous aerobic fermentation apparatus, including a vertical cylindrical fermentor vessel, an internal cooling apparatus in combination therewith, said cooling apparatus comprising,

a. a top circular header pipe disposed horizontally within an upper section of the fermentor vessel;

b. a bottom circular header pipe, aligned axially with and having substantially the same mean diameter as the top header pipe, disposed horizontally within a lower section of the fermentor vessel;

c. a plurality of connector pipes, disposed vertically in a spaced relationship and severally attaching the bottom circular header pipe to the top circular header pipe, being arranged in parallel rows along the circumference described by the circular header pipes in sufficient number to provide a total crosssection area substantially equal to the cross-section area of either of the header pipes;

d. an inlet pipe, extending horizontally through the fermentor vessel wall in sealed relation thereto in the plane of the bottom header pipe and attached at one end to the header pipe;

e. an exit pipe, attached at one end to the top circular header pipe and extending horizontally therefrom and passing through the fermentor vessel wall in sealed relation therewith; and

f. pumping and compressing means located externally of the fermentor vessel and attached externally thereof to the extended ends of both the inlet pipe and the exit pipe, in order to provide a continuously flowing system for containment of coolant liquid and vapor phases, whereby efficient indirect heat exchange is effected with a fermentation liquor contained in the sections of the fermentor vessel and surrounding the internal cooling apparatus, so that a substantially constant temperature is maintained within the fermentation liquor throughout the course of the aerobic fermentation processing; and said fermentor vessel comprising,

1. an air inlet and outlet means attached to the top section;

2. an agitator means located directly below the bottom circular header pipe;

3. an air sparger means attached to the air entry means within the bottom section and situated directly beneath the agitator means;

4. a nutrient inlet means attached to the top section;

5. a fermentation broth outlet means attached to the bottom section; and

6. two vertical baffle means located along the interior wall of the vessel on either side of the internal cooling apparatus.

2. The apparatus of claim 1 wherein the top and bottom circular header pipes are disposed within a fermentor vessel having a capacity of about 50,000 gallons and are fashioned from pipe having a diameter of about two feet to describe a circle whose outside diameter is about 16 feet.

3. The apparatus of claim 2 wherein the vertical connector pipes are fashioned from pipe having a diameter of about 1 inch and are arranged in two parallel rows about the circumference of the circular header pipes.

22);? UNITED STA'IES PAN-3N1 OFFICE CERTIFICATE OF CORRECTION Patent No. 3,8h7fl50 I Dated November '12, $7 k John A. Ridgway; Jr., Terry A. Lappin, Benny Moses Benjamin, Inventor(s) Joseph B. Corns and Cavit Akin It is oertified that error appears in the aboveddcntified paten't and that said Letters Patent are hereby corrected as shown below:

Column 3, line 25, "wtaer" should be water Column 6, line 1, "flter" should be filter Column 8, line v +9, "Typtophane" should be Tryptophane Column '9, line 25, "denstiy" should be density ----3 v line 65, "solugin" should be solution Signed and sealed this 4th day of March 1975.

(SEAL) Attest: v

' I C. MARSHALL DANN RUTH C. IlASON Commissioner of Patents I Attesting Officer and Trademarks 

1. IN A CONTINUOUS AEROBIC FERMENTATION APPARATUS, INCLUDING A VERTICAL CYLINDRICAL FERMENTOR VESSEL, AN INTERNAL COOLING APPARATUS IN COMBINATION THEREWITH, SAID COOLING APPARATUS COMPRISING, A. A TOP CIRCULAR HEADER PIPE DISPOSE HORIZONTALLY WITHIN AN UPPER SECTION OF THE FERMENTOR VESSEL; B. A BOTTOM CIRCULAR HEADER PIPER, ALIGNED AXIALLY WITH AND HAVING SUBTANTIALLY THE SAME MEANS DIAMETER AS THE TOP HEADER PIPE, DISPOSED HORIZONTALLY WITHIN LOWER SECTION OF THE FERMENTOR VESSEL; C. A PLURALITY OF CONNECTOR PIPES, DISPOSED VERTICALLY IN A SPACE RELATIONSHIP AND SEVERALLY ATTACHING THE BOTTOM CIRCULAR HEADER PIPE TO THE TOP CIRCULAR HEADER PIPE, BEING ARRANGED IN PARALLEL ROWS ALONG THE CIRCUMFERENCE DESCRIBED BY THE CIRCULAR HEADER PIPES IN SUFFICIENT NUMBER TO PROVIDE A TOTAL CROSS-SECTION AREA SUBSTANTIALLY EQUAL TO THE CROSS-SECTION AREA OF EITHER OF THE HEADER PIPES; D. AN INLET PIPE, EXTENDING HORIZONTALLY THROUGH THE FERMENTOR VESSEL WALL IN SEALED RELATION THERETO IN THE PLANE OF THE BOTTOM HEADER PIPE AND ATTACHED AT ONE END TO THE HEADER PIPE; E. AN EXIT PIPE, ATTACHED AT ONE END TO THE TOP CIRCULAR HEADER PIPE AND EXTENDING HORZONTALLY THEREFROM AND PASSING THROUGH THE FERMENTOR VESSEL WALL IN SEALED RELATION THEREWITH; AND F. PUMPING AND COMPRESSING MEANS LOCATED EXTERNALLY OF THE FERMENTOR VESSEL AND ATTACHED EXTERNALL THEREOF TO THE EXTENDED ENDS OF BOTH THE INLET PIPE AND THE EXIT PIPE, IN ORDER TO PROVIDE A CONTINUOUSLY FLOWING SYSTEM FOR CONTAINMMENT OF COOLANT LIQUID AND VAPOR PHASES, WHEREBY EFFICIENT INDIRECT HEAT EXCHANGE IS EFFECTED WITH A FERMENTATION LIQUOR CONTAINED IN THE SECTIONS OF THE FERMENTOR VESSEL AND SURROUND THE INTERNAL COOLING APPARATUS, SO THAT A SUBSTANTIALLY CONSTANT TEMPERATURE IS MAINTAINED WITHIN THE FERMENTATION LIQUOR THROUGHOUT THE COURSE OF THE AEROBIC FEMENTATION PROCESSING; AND SAID FERMENTOR VESSEL COMPRISING,
 1. AN AIR INLET AND OUTLET MEANS ATTACHED TO THE TOP SECTION;
 2. an agitator means located directly below the bottom circular header pipe;
 2. The apparatus of claim 1 wherein the top and bottom circular header pipes are disposed within a fermentor vessel having a capacity of about 50,000 gallons and are fashioned from pipe having a diameter of about two feet to describe a circle whose outside diameter is about 16 feet.
 2. A AN AGITATOR MEANS LOCATED DIRECTLY BELOW THE BOTTOM CIRCULAR HEADER PIPE;
 3. an air sparger means attached to the air entry means within the bottom section and situated directly beneath the agitator means;
 3. AN AIR SPARGER MEANS ATTACHED TO THE AIR ENTRY MEANS WITHIN THE BOTTOM SECTION AND SITUATED DIRECTLY BENEATH THE AGITATOR MEANS;
 3. The apparatus of claim 2 wherein the vertical connector pipes are fashioned from pipe having a diameter of about 1 inch and are arranged in two parallel rows about the circumference of the circular header pipes.
 4. a nutrient inlet means attached to the top section;
 4. A NUTRIENT INLET MEANS ATTACHED TO TOP SECTION;
 5. A FERMENTATION BROTH OUTLET MEANS ATTACHED TO THE BOTTTOM SECTION; AND
 5. a fermentation broth outlet means attached to the bottom section; and
 6. two vertical baffle means located along the interior wall of the vessel on either side of the internal cooling apparatus.
 6. TWO VERTICAL BAFFLE MEANS LOCATED ALONG THE INTERIOR WALL OF THE VESSEL ON EITHER SIDE OF THE INTERNAL COOLING APPARATUS. 